Optimizing Oligonucleotide Drug Products: Strategies for Formulation, Lyophilization, and Fill Finish Processes

Imagine if you could create a targeted, highly effective, and life-saving treatment to a disease once thought untreatable and incurable with few to no side effects. That is what the biotherapeutic – oligos – is offering.

Oligonucleotides (or “oligos”) are single- or double-stranded synthetic nucleic acid polymers designed to bind to RNA or DNA and can be created to target pre-mRNA, mRNA, or non-coding RNA to induce degradation, modulate splicing events, or interfere with protein translation. They are rapidly gaining momentum as a highly targeted treatment capable of preventing or modulating the expression of nearly any gene and have quickly become the up-and-coming solution to complex diseases.

Despite the promise that oligonucleotides hold in unlocking new drug therapies for previously incurable and untreatable diseases, challenges in manufacturing these new drugs can prevent them from making it to the market. Therefore, while this new type of biotherapeutic offers incredible potential, it is essential to address the challenges in manufacturing and ensure that these life-saving treatments reach the patients who need them.

Therapeutic oligonucleotides often face challenges that complicate the drug product development or fill finish process. These include:

  • Temperature sensitivity – most oligonucleotides are unstable at room temperature and are required to be frozen or lyophilized to maintain their shelf life
  • Shear sensitivity – some oligonucleotides are also shear-sensitive, especially those packaged in lipid nanoparticles (LNPs)
  • Risk of degradation from ribonucleases – unprotected RNA can be quickly destroyed by ribonucleases (or “RNases) that naturally in the environment and on people
  • Being highly expensive to produce or difficult to procure – oligonucleotides are a high-value drug substance where purchasing excess material is difficult and expensive. Reducing drug product loss during the fill finish process is critical to supplying clinical trials or commercial products to patients.

In this blog, we discuss this drug product development and fill finish process for oligonucleotides and offer our solutions to the challenges listed above.

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Download our white paper: From Bench to Bedside: The Journey of Oligonucleotide Drug Development and Sterile Filling

Formulation development of oligos

Developing the drug product formulation for an oligo therapy is critical to extending shelf life, increasing efficacy, and successfully transferring the manufacturing process to a GMP setting.

The first step to developing the optimal formulation for your drug product is to determine a buffer composition and optimal pH for your drug. Your CDMO (Contract Development and Manufacturing Organization) will offer their suggestions for a buffer composition and pH before performing studies to optimize each. These studies will involve preparing several formulations at different excipient and buffer concentrations and pH ranges then placing samples from each on accelerated stability for approximately three months to determine which formulation performs the best. The formulation that demonstrates the greatest stability will be recorded and tested further to improve the stability even more.

After establishing a potential formulation, your CDMO will evaluate any product sensitivities that can affect the fill finish process. There are several tests that your CDMO may offer for your drug product:

  • Freeze-thaw testing – this testing is primarily performed for frozen drug product, and it determines if the formulation can withstand typical shipping conditions 
  • Shear testing – this testing determines the optimal pumps, pump speeds, and process requirements to use in fill finish for shear sensitive drug products. Shear testing involves multiple tests – mixing studies, fill studies, and filter studies – to determine requirements and settings. 
  • Syringeability study – this study is often performed for suspensions, including LNP products to ensure the formulation is optimized for consistent, accurate, and complete dosing in the clinic. 

Your CDMO will perform several other studies to optimize your formulation process and ensure your drug product can withstand a fill finish process.  

  • Filterability study – filterability studies are performed to determine the optimal filter membrane and size to use in sterile filtration. After a filter is decided on, the filtering process will be validated. Validation of the filtering process is a regulatory requirement for all GMP manufactured drug product when commercially available. This is typically done immediately prior to the product going commercial as the formulation and filter need to be set prior to taking on this activity.
  • Component compatibility study – the components used in manufacturing (e.g. tubing, connectors, bio process bags, etc.) can affect the final drug product formulation. These materials can absorb the API or excipients, or leach chemicals into the drug product formulation. Component compatibility studies determine if any selected materials that will be used in the fill finish process will have a detrimental effect on the drug product, and allow the CDMO to offer substitutions or process adjustments to prevent degradation.  
  • Hold time studies – These studies determine if the drug product can withstand the conditions it will be under during the fill finish process. This is required prior to going commercial and is typically performed with the PV batch production once phase III is complete and the formulation and dosing are set. This will advise the CDMO of the formulation and processing constraints on temperature during production, packaging, and release to the patient. 
Figure 1. API Concentration vs. Time (in hours) from an example hold time study. If the drug product (DP) remains within the acceptable range for the entire hold time study (DP 1), then the process and formulation do not require any modifications. If the drug product falls out of acceptable range late in the hold time study (DP 2), then changes to the process and formulation should be explored, but the manufacturing process may be able to be shortened. If the drug product falls out of range early in the study (DP 3), then changes to the formulation or process must be made for the product to be manufacturable.

For an in-depth look at what these studies involve and how they are performed, check out our white paper:

From Bench to Bedside: The Journey of Oligonucleotide Drug Development and Sterile Filling

Lyophilization development of oligos

As mentioned earlier, many oligo therapeutics are temperature sensitive. One solution to extend shelf-life and protect the product is to freeze-dry the drug product via lyophilization. 

Creating a lyophilization cycle is not a trial-and-error process. A typical lyo cycle development project will include the following steps: 

Figure 2. Berkshire Sterile’s lab-scale IMA lyophilizer is used for lyo cycle development and optimization and features a weigh cell to track the drying rate with time.

Determining thermal characterizations

Understanding the thermal characterizations of the API and excipients in a drug product formulation is key to developing an optimal lyophilization cycle. The CDMO may perform studies and testing in-house or outsource this work to determine the:

  • Glass transition temperature 
  • Eutectic temperature 
  • Collapse temperature 
  • Re-crystallization temperature 

for the oligo and each excipient. The CDMO will also analyze the drug product formulation and classify the oligo and each excipient as either a crystalline or amorphous solution. 

Once these thermal characterizations are determined, an initial lyophilization cycle can be created. 

Optimizing solvent removal

During this process, the initial lyophilization cycle is tested and the drug product is visually inspected for common issues that can occur in lyophilization, including, but not limited to:

  • Cake collapse
  • Meltback
  • Drops on the vial wall
  • Detached cake
  • Inconsistent mass
  • Cake shrinkage
  • Puffing
  • A rigid and impermeable top layer

Each of these problems can be solved by adjusting the lyophilization cycle. Once adjustments are made and the lyophilization cycle is consistently producing quality cakes, the process can be optimized even further to reduce cycle time.

During this process, the CDMO will look for pressure drops inside the lyophilizer at the beginning and end of primary drying to determine if this cycle can be reduced. They will also analyze the drug product and determine if adding an annealing phase would be beneficial to reducing drying time in the secondary drying phase. Optimizing the lyo cycle time will be beneficial to reducing manufacturing time and limiting energy waste.

Figure 2. Berkshire Sterile’s lab-scale IMA lyophilizer is used for lyo cycle development and optimization and features a weigh cell to track the drying rate with time.

Performing stability studies

Once an effective and optimized lyo cycle is developed, the CDMO will perform stability studies to estimate the shelf-life of the cake and determine ideal storage conditions for the drug product. Typically, this is performed by placing samples on accelerated stability for a minimum of three to four months. These stability studies will not be able to be used to established estimated stability for GMP product, but it will advise the CDMO and client on formulation, process, and storage requirements and ideal conditions. Once a full-scale engineering run of the sponsor’s fill finish project is performed, samples can be pulled for nominal and accelerate ICH stability studies to establish an estimated and a true expiration that will be accepted by regulatory agencies.

Determining process robustness and tech transfer to an industrial lyophilizer

Once a lyophilization cycle has been established, the next step will be to transfer the process to a cGMP lyophilizer that will be used in manufacturing. The efficacy of the lyo cycle is subject to the heat transfer between the shelf and the vials. Each lyophilizer will cool differently, and there are also temperature differences between the edge and center of the lyophilizer that will affect drying times. The larger the lyophilizer or the higher the percent capacity of the lyophilizer is, the longer the cycles will need to be to ensure all product is cooled and heated to the appropriate temperature for the necessary amount of time at each phase of the process.

If the lyophilization process is being transferred internally at a CDMO, the transfer is often more efficient and streamlined, since the CDMO will have a deeper understanding the of cycle requirements and adjustments that need to be performed to transfer a cycle developed on their lab-scale lyo to a cGMP industrial lyo on their GMP filling lines.

For a more detailed description of the lyo cycle development process, check out our white paper:

From Bench to Bedside: The Journey of Oligonucleotide Drug Development and Sterile Filling

Formulation and filling of oligos

The process for formulating, filtering, and filling oligo therapeutics is not unique. However, these products often do have several product sensitivities that add challenges to the process. Where applicable, these can include:

In this section, we will briefly cover changes to the process that your CDMO will likely offer to protect your drug product during formulation, filtration, and filling.

Protecting temperature-sensitive drug product

All oligo therapeutics that we filled at Berkshire Sterile Manufacturing were temperature sensitive. Most oligos are unstable in aqueous solution and benefit from remaining frozen or undergoing lyophilization to extend their shelf-life. Unfortunately, formulation, filtering, and filling are performed in cleanrooms at controlled room temperature, and leaving these sensitive drug products at these temperatures can cause considerable damage.

Fortunately, there are several strategies to keep product cool or limit its exposure to heat:

  • Using jacketed mixing vessels – the CDMO can formulate in, filter into, and fill from a mixing vessel with a water jacket. The ones used at Berkshire Sterile, for example, are hooked up to a controlled water bath and circulate chilled water (6 – 15°C) to keep the drug product cool throughout the fill finish process
  • Thawing in a 2-8°C temperature-controlled unit (TCU) – if the drug product or drug substance arrives as a frozen solution, it can be thawed in a cooler. If the drug substance arrives as a powder, then it will be removed from the freezer directly before formulation activities begin
  • Expediting activities – If the drug product is exceptionally temperature sensitive, many processes can be expedited. Filling can occur directly after formulation, instead of performing an overnight hold, and visual inspection, which occurs after product is filled and before product is frozen, can be expedited or be performed “on-the-line”. In the latter situation, operators will immediately remove filled trays of drug product to the visual inspection suite for visual inspection activities. As the trays are inspected, they will be moved into the freezer. 

If your drug product is temperature sensitive, it’s important to work with a CDMO that can accommodate this sensitivity and has had practice in managing it.

Figure 3. Sterile filtering using jacketed vessels. Jacketed vessels circulate cool water (6 – 15°C) around the outside of the glass mixing vessels to keep the drug product chilled. These are often used during formulation, filtration, and filling to limit heat exposure.

Protecting shear-sensitive drug product 

Many oligonucleotide therapies, especially those packaged in protect lipid nanoparticles (LNPs) are exceptionally shear-sensitive. Mixing and pumping motions that the drug product is subjected to during formulation and filling can cause protein aggregation, compromising the product’s function and efficacy. Performing mixing studies, filtration studies, and filling studies are critical to understanding the extent of the sensitivity and the options available to the sponsor. They will also identify the optimal pump type and mixing and pumping speeds to use in the fill finish process. However, here are some solutions to limiting shear during formulation, filtration, and filling:

  • Reducing mixing and pumping speed – reducing the speed of these motions will reduce shear. These should be optimized, however, to ensure the process is time-efficient while preventing degradation 
  • Using a low shear vertical blender – for large formulations, using a low shear vertical blender will protect the drug product during formulation 
  • Changing to a peristaltic or piston pump – many will say that a piston pump creates more shear than a peristaltic pump because they compress the fluid where peristaltic pumps squeeze the fluid. However, the effect of the pump can be product specific, so it is worth exploring another pump option to determine if it would be a better fit. 
  • Using a time-over-pressure filler – this is a unique filling mechanism that does not have a pump. Instead, the drug product is pressurized to a low pressure (typically 1 psi), and dispensing is controlled by a time-operated actuator that opens and closes an opening to the filling needle. This type of filling is offered at Berkshire Sterile on their low loss filler. 
Figure 4. Berkshire Sterile’s low loss fill process set up (pictured) does not use a pump and is ideal for filling extremely shear sensitive drug products

Protecting RNA APIs 

Ribonucleases (or “RNases”) exist naturally in the environment. When they are in the presence of unprotected RNA, it will quickly cleave and destroy it. This poses a significant problem for unprotected RNA APIs. While most sponsors will typically modify their drug to offer some protection from RNases (e.g., enclosing their oligonucleotide in an LNP), some do not protect the drug substance, and it is up to the CDMO to take extra precaution to clean and prevent contamination that could lead to its degradation. 

The solution to protecting RNA APIs is to use RNase-free equipment and water for any activity involving the RNA drug substance. Water for Injection (WFI) is considered RNase-free, too. 

The CDMO should also keep a clean and dedicated area for this work and be mindful of their environment and activities to avoid contamination. 

Finally, it may be beneficial to keep the drug product cool or frozen since the rate of degradation decreases when the temperature of the product drops. 

Reducing drug product loss 

Developing and manufacturing oligonucleotides can be exceedingly expensive, especially for those sponsors pursuing personalized medicines or treatments for rare diseases with a small patient population. Fortunately, there are many practices that your CDMO can implement to reduce product loss: 

  • Filtering into bioprocess bags versus other vessels 
  • Using fewer and smaller filters 
  • Reducing line purge requirements 
  • Performing non-destructive weight checks 
  • Performing fewer destructive weigh checks 
  • Lifting the bulk drug product at the end of a fill to allow for more product transfer

And there is a new method of filling at Berkshire Sterile Manufacturing that significantly limits product loss for small batch filling of drug product. The low loss fill process at their site limits product loss to less than 30 mL of drug product and is ideal for low volume drug product lots (1.2 liter or less). 

Figure 5. Sterile filling in Berkshire Sterile’s manual isolator line using their low loss fill process. 

To get an in-depth guide to reducing drug product loss in fill finish, download our free white paper:

Minimizing volume losses in small-scale aseptic fill finish for high-value drug products

Conclusion

The fill finish process for oligonucleotide drug products is a critical stage in the manufacturing of these specialized medicines. It involves a series of complex operations that are designed to ensure the safe and effective delivery of the drug to patients. While the process can be complicated by sensitivities and reducing drug product loss, careful planning can help optimize the process and get it ready for success in a GMP setting. 

This blog has provided an overview of the key considerations involved in the fill finish process for oligonucleotide drug products, including the importance of formulation development, the process of developing an optimal lyophilization cycle, and techniques to protect the product in manufacturing and limit drug product loss. 

Overall, the fill finish process is a critical component of oligonucleotide drug product manufacturing that requires a high degree of expertise and attention to detail. By leveraging the best practices outlined in this white paper, manufacturers can ensure these innovative drug products are delivered to sponsors, their clinics, and to patients. 

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